Annealing Temperature
Definition
The reduced temperature (50-65°C) at which primers hybridise to single-stranded template. Determined by primer Tm; set to balance specificity against yield.
- Typical range
- 50-65°C
- Determined by
- Primer melting temperature (Tm)
- Function
- Primer-template hybridisation
- Design tradeoff
- Specificity versus yield
Common questions
What happens if the annealing temperature is set too low for the primers used?+
Primers can bind to partially mismatched sequences elsewhere in the template, producing non-specific amplification products and off-target bands that can complicate or invalidate downstream STR profile interpretation.
What happens if it is set too high?+
Primer binding becomes too weak and inefficient, reducing amplification yield and potentially causing allele or locus dropout, which is particularly damaging when working with low-template or degraded forensic samples.
Related terms
- A-Addition
- The non-templated addition of a single adenine nucleotide to the 3' end of PCR products by Taq polymerase, exploited in forensic STR...
- AmpliTaq Gold
- A chemically modified hot-start form of Taq polymerase (Applied Biosystems) that is inactive at room temperature and requires a 10-11 minute activation...
- Denaturation
- The separation of double-stranded DNA into two single strands by disrupting the hydrogen bonds between base pairs. In PCR, denaturation is achieved...
- Extension
- The PCR step at ~72°C at which thermostable DNA polymerase synthesises a new complementary strand from each primer, using dNTPs in the...
- Hot-Start Polymerase
- Any Taq formulation (antibody-mediated, chemically modified, or aptamer-blocked) that is inactive below ~60°C, preventing non-specific amplification during reaction setup.
- Melting Temperature (Tm)
- The temperature at which half of a primer-template duplex population is dissociated. Depends on primer length, GC content, and nearest-neighbour stacking interactions.
- Negative Amplification Control
- A no-template reaction well run alongside case samples; any amplified peak indicates reagent contamination or analyst-introduced contamination during setup.
- Uni-Directional Workflow
- The physical principle governing forensic DNA laboratory layout: personnel and materials move from pre-PCR to post-PCR zones only, preventing high-copy-number amplified products...